Quantitation of viruses by light scattering

ABSTRACT

A method for determining the number or concentration of virus particles in a sample by use of a light scattering detector. The method may be used to quantitate purified virus preparations or virus samples containing contaminants, including ultraviolet light-absorbing contaminants, such as proteins. The method is useful for quantitation of viruses for use in gene therapy, oncolytic viruses for tumor cell lysis and virus-based vaccines.

FIELD OF THE INVENTION

The present invention relates to virus quantitation. More specifically,the invention relates to quantitation of viruses by measuring scatteredlight.

DESCRIPTION OF THE RELATED ART

Determination of virus concentration is important in, among otherthings, quantitation of viral vectors for use in gene therapy,quantitation of oncolytic viruses and quantitation of virus-basedvaccine compositions. The quantitation of viruses for use in genetherapy with accuracy and precision is critical to ensure adequatecomparability of data obtained in various intra- and inter-institutionalstudies, as well as to ensure comparability between virus preparationsused for preclinical and clinical studies (Mittereder et al., J. Virol.70:7498-7509, 1996). The quantitation of viruses that lyse tumor cells(oncolytic viruses) is also important in determining the correct dosage.In addition, quantitation of virus-based vaccines is important forsafety and efficacy of administration of these compositions.

Examples of oncolytic viruses include mutated adenovirus (Heise et al.,Nat. Med. 3:639-645, 1997), mutated vaccinia virus (Gnant et al., CancerRes. 59:3396-3403, 1999) and mutated reovirus (Coffey et al., Science282:1332-1334, 1998). Examples of viral vectors for use in gene therapyinclude mutated vaccinia virus (Lattime et al., Semin. Oncol. 23:88-100,1996), mutated herpes simplex virus (Toda et al., Hum. Gene Ther.9:2177-2185, 1998), mutated adenovirus (U.S. Pat. No. 5,698,443) andmutated retroviruses (Anderson, Nature 392(Suppl.):25-30, 1998).

As early as the 1960s and 1970s, light scattering measurements were usedto study the assembly and aggregation of viral components and viralparticles (Smith et al., Biochemistry 6:2457-2464, 1967; Cummins et al.,Biophys. J. 9:518-546, 1969; Camerini-Otero et al., Biochemistry26:960-970, 1974). Diffusion coefficients, molecular weights andparticle dimensions of viruses and viral components have all beenstudied with light scattering techniques. These studies have emphasizedthe variation of light scattering per virus particle, depending on thestate of aggregation, association, dissociation, etc., of the virusparticles. Modern light scattering detectors are designed to permitcharacterization of the size distributions of molecules and particles,including viruses, using an auxiliary detector (e.g., ultraviolet lightabsorbance detector or refractive index detector) as the concentrationdetector.

U.S. Pat. No. 5,837,520 to Shabram et al. discloses and claims a methodfor determining the number of intact virus particles in a sample bymonitoring the ultraviolet absorbance of the effluent from a column ofan anion exchange resin and comparing that absorbance to a standardcurve that is prepared with virus suspensions of known concentrations.This method, with measurement of light absorbance at 260 nm and 280 nm,is used by Shabram et al. (Hum. Gene Ther. 8:453-465, 1997) toquantitate adenovirus in suspensions.

Publications on light scattering in the context of virus quantitationemphasize the interference by light scattering with ultraviolet lightabsorbance measurements (Maizel et al., Virology 36:115-125, 1968;Tikhonenko et al., Mol. Biol. (Moscow) 12:393-395, 1978; Mittereder etal. supra). Tsoka et al. (Biotechnol. Bioeng. 63:290-297, 1999) usedynamic light scattering to detect distributions of particle sizes insuspensions of virus-like particles, and teach that it is necessary toadd antibodies to a suspension of virus-like particles in order toinduce a change in particle size that can then be measured by dynamiclight scattering.

Dynamic light scattering measurements are distinct from static lightscattering measurements. Typically, dynamic light scattering (also knownas photon correlation spectroscopy) is an optical method used to studythe Brownian motion of particles in solution. Measurements are taken todetect fluctuations in the intensity of light scattered by a sample, attime points on a scale related to the time taken for a particle todiffuse a distance comparable to the wavelength of the light scattered.See Tsoka et al. In contrast, static light scattering is not based onfluctuations in intensity over time, and is not directed to detectingBrownian motion or diffusion rates of particles.

Bistocchi et al. (Tumori 63:525-534, 1977) describe quantitation ofmurine mammary tumor virus (muMTV) in mouse milk, and refer to theirvirus quantitation as having been done by “light scattering.” However,this reference does not actually describe the use of a light scatteringdetector for quantitating viruses, but instead describes the measurementof ultraviolet light absorbance at 260 nm, which is also referred to bythose authors as optical density. It is the increase in optical density(i.e. the decrease in transmitted light) that these authors refer to as“light scattering.” Light absorbance and light scattering are distinctphenomena: the optical density values of the samples of Bistocchi et al.include contributions from light scattering by the virus particles(which decreases the amount of light transmitted through the sample), aswell as contributions from light absorbance at 260 nm by milk proteinsand by viral nucleic acids. The authors took into account the expectedlight absorbance by milk proteins, based on the protein content of themilk as determined by the method of Lowry et al. (J. Biol. Chem.193:265-275, 1951). However, they apparently assumed that aftercorrecting for the light absorbance due to milk proteins, the resultwould be a measure of light scattering by virus particles. They did nottake into account the light absorbance at 260 nm due to viral nucleicacids. In any case, this reference does not actually report quantitationof a virus by light scattering, but instead reports quantitation of avirus by an adjusted or corrected ultraviolet light absorbancemeasurement.

An important difference between an absorbance measurement and a lightscattering measurement is that the detector for measuring absorbancemust be placed on the side of the sample opposite to the light source,along the axis of illumination, where it measures the decrease in lighttransmitted through the sample, as done by Bistocchi et al. On the otherhand, a detector for measuring scattered light is placed away from theaxis of illumination, for example at 90 degrees to that axis, to measurethe increase in light that is scattered by the sample at a non-zeroangle to the incident beam. A second difference between absorbance andscattering measurements stems from the necessity to employ a wavelengththat is specifically absorbed by the sample in the former method (e.g.ultraviolet light with a wavelength of 260 or 280 nm), whereaswavelengths that are not absorbed by the sample are preferred in thelatter method (e.g. visible light with a wavelength of 690 nm from adiode laser, 632.8 nm from a helium-neon laser or 488 nm from anargon-ion laser).

There is an ongoing need for more accurate methods for measuring virusconcentrations, particularly for adenovirus, for which no widelyaccepted standard method is known. The present invention addresses thisneed.

SUMMARY OF THE INVENTION

The present invention provides a method for determining the number orconcentration of virus particles in a sample, including the steps of:measuring the amount of light scattered by the virus particles; andcomparing the amount of light scattered to one or more known lightscattering values correlated with one or more known concentrations ofthe virus. The comparing step may include a standard light scatteringcurve including several data points, wherein the data points are basedon the light scattering values and the concentrations of the virus. Themethod may further include the step of purifying the virus particlesusing a fractionation system prior to the measuring step. Thefractionation system may include a chromatographic medium, such as, forexample, an ion-exchange medium, a size-exclusion medium, an affinitymedium, and the like. The fractionation system may include one or morecomponents such as a chromatography column, a countercurrentdistribution apparatus, a two-phase system, a gradient, or a centrifuge.The virus may be, for example, an adenovirus, human herpesvirus, humanpapilloma virus, adeno-associated virus, flavivirus, dengue virus,Japanese encephalitis virus, human T-cell lymphotrophic virus, hepatitisvirus, human immunodeficiency virus (HIV), cytomegalovirus (CMV),Epstein-Barr virus, reovirus, vaccinia virus, parvovirus, felineleukemia virus, cauliflower mosaic virus and tomato bushy stunt virus.

In another embodiment, the invention provides a system for quantitationof virus particles including a light source adapted for directing lightalong a light path, a sample within the light path, a detectorpositioned to detect light scattered at an angle to the light path, anda recorder in communication with the detector, wherein the sample mayinclude a quantity of particles of a virus, and wherein a portion of thelight may be scattered from the path at the angle by the virusparticles, and wherein the detector detects the light scattered at theangle to produce a signal that is a function of the quantity of virusparticles, and wherein the signal may be communicated to the recorderand converted to a value indicating the quantity of the virus particles.The detector may be selected from the group consisting of a multi-angledetector, a dual-angle detector, and a single-angle detector. The systemmay further include a fractionation system in communication therewith,wherein the fractionation system receives a pre-sample including thevirus particles and other components, and wherein the fractionationsystem separates the virus particles from the other components. Thevirus may be, for example, an adenovirus, human herpesvirus, humanpapilloma virus, adeno-associated virus, flavivirus, dengue virus,Japanese encephalitis virus, human T-cell lymphotrophic virus, hepatitisvirus, human immunodeficiency virus (HIV), cytomegalovirus (CMV),Epstein-Barr virus, reovirus, vaccinia virus, parvovirus, felineleukemia virus, cauliflower mosaic virus and tomato bushy stunt virus.The quantity measured by the system of this aspect of the invention maybe a concentration of virus particles per unit volume of a liquidsample. For example, such concentration may be between about 10⁸ and10¹² virus particles/mL. Likewise, the quantity measured by the systemof this aspect of the invention may be a number of virus particles inthe sample. For example, such number may be between about 10₈ and 10¹⁰virus particles.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing detection of 4.4×10⁹ adenovirus particles byion-exchange chromatography using light scattering at 90° and absorbanceat 260 nm.

FIG. 2A is a graph showing standard curves for quantitation ofadenovirus detected by light scattering at 90° and by absorbance at 260nm.

FIG. 2B is a graph showing a standard curve for quantitation ofadenovirus detected by absorbance at 260 nm, on an expanded scalerelative to FIG. 2A.

FIG. 3 is a graph showing light scattering at two angles and absorbancemeasured at 260 nm for quantitation of 5.5×10⁸ adenovirus particles.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention includes the observation that the use of a lightscattering detector as an alternative to an ultraviolet absorbancedetector confers an unexpected increase in sensitivity and specificityof virus quantitation. Suitable light scattering detectors for use withthis invention are multi-angle laser light scattering detectors (e.g.DAWN DSP or MiniDAWN detectors manufactured by Wyatt Technology Corp. ofSanta Barbara, Calif.), dual-angle light scattering detectors orsingle-angle light scattering detectors, as illustrated in Examples 1through 6.

This method can be used to quantitate any virus for which a suspensionof homogeneous, purified virus particles is available at a knownconcentration, permitting calibration of the light scatteringmeasurements with dilutions of the standardized suspension of thatvirus. In a preferred embodiment, virus particles are purified using anyconventional fractionation system, such as a system that includes achromatographic column. The chromatographic column may contain anion-exchange medium (cationic or anionic), a size-exclusion medium, anaffinity resin, or any other medium or resin capable of removing,retaining or retarding the movement of contaminants (molecules otherthan the virus particles of interest) so that virus particles arepresent in a substantially purified form in at least a portion of thecolumn effluent. Many types of suitable separation media are availablefrom many commercial sources, including Amersham-Pharmacia Biotech(Piscataway, N.J.), Sigma Chemical Co. (St. Louis, Mo.) and Bio-RadLaboratories (Hercules, Calif.). The fractionation system may alsoinclude a two-phase system comprising aqueous solutions of dextran andpoly(ethylene glycol), with or without a countercurrent distributionapparatus to facilitate separation of the two phases, a zonal orcontinuous-flow centrifuge, an ultracentrifuge employing, for example,density gradients, whether continuous or step gradients, and,additionally or alternatively, the fractionation system may include oneor more size-selective membranes with appropriate molecular weightlimits to separate the virus particles from accompanying molecules orcell debris, or both.

In a preferred embodiment, the fractionation system binds non-viralcomponents and the virus particles appear in the eluate. In analternative embodiment, the virus particles are retained in the columnand the contaminants appear in the eluate. This is accomplished, forexample, by using an affinity adsorbent to which an antibody havingaffinity for a viral surface protein is bound. The bound virus is theneluted using, for example, a peptide that competes with the virus forbinding to the adsorbent.

Although the quantitation of purified virus preparations is preferred,the present method can also be used to quantitate virus withoutchromatographic separation, as demonstrated by the independence of themeasurement of virus concentration on the concentration of contaminatingbovine or human serum albumin, even when the contaminating proteinrepresents more that 75%, 90%, 95%, or 99% of the protein present in thevirus suspension. Light scattering can also be used to measure virusconcentrations in virus suspensions containing polymers including, butnot limited to, poly(ethylene)glycol and dextran. For accuratequantitation of virus particles by light scattering measurements in thepresence of polymers, such polymers must not be sufficientlyconcentrated that they induce aggregation of the virus particles.

The present method may be used to quantitate viral vectors for use ingene therapy applications such as adenovirus and herpesviruses, forquantitation of oncolytic viruses such as ONYX-015 (U.S. Pat. Nos.5,677,178 and 5,846,945), and for quantitation of virus-based vaccines,such as those for poliovirus, varicella-zoster virus, measles virus, andthe like.

Although the use of light scattering for quantitation of adenovirus isdescribed herein, this method may be used to quantitate any desiredvirus present in a sample including, for example, adeno-associatedvirus, human herpesvirus, human papilloma virus, pathogenic humanflaviviruses such as dengue virus and Japanese encephalitis virus, humanT-cell lymphotrophic viruses (HTLV-I and HTLV-II), hepatitis viruses A,B and C; human immunodeficiency viruses (HIV-1 and HIV-2),cytomegalovirus (CMV), Epstein-Barr virus (EBV), reovirus, vacciniavirus, canine parvovirus, feline leukemia virus, plant viruses such ascauliflower mosaic virus and tomato bushy stunt virus (TBSV), and thelike. Viruses may be naturally occurring, genetically engineered, orotherwise modified; likewise the viruses may be virulent, attenuated,tropism-restricted, or killed, depending on the intended use thereof.Further, some embodiments of the present invention are likewise suitablefor quantitation of virus-like particles as well as virus particles.

Static light scattering measurements thus offer high sensitivity ofvirus detection, and precision of quantitation across a wide range ofvirus particle concentration or absolute numbers of virus particles.These ranges may vary depending on the nature of the light scatteringsystem and other software, hardware, or devices used in connection withthe light scattering detector. For example, such concentration may bebetween about 10⁸ and 10¹² virus particles/mL. Likewise, the quantitymeasured by the system of this aspect of the invention may be a numberof virus particles in the sample. For example, such number may bebetween about 10⁸ and 10¹⁰ virus particles.

EXAMPLE 1 Determination of Adenovirus Concentration by Light Scattering

Portions of a suspension of adenovirus (ONYX-015, Onyx Pharmaceuticals,Richmond, Calif.) containing 1.1×10¹² particles/mL were diluted 5-, 10-or 20-fold. Injections of 10, 20 or 40 μL were chromatographed on a 1-mLResource Q anion-exchange column (Amersham-Pharmacia Biotech,Piscataway, N.J.), essentially as described in U.S. Pat. No. 5,837,520,the entire contents of which are hereby incorporated by reference,except that the effluent from the UV2000 ultraviolet light absorbancedetector (Thermo Separation Products, San Jose, Calif.), which wasprogrammed to measure absorbances at 260 nm and at 280 nm, was connectedto a multi-angle light scattering detector with a 690-nm laser lightsource (MiniDAWN, Wyatt Technology Corp., Santa Barbara, Calif.). The 0-to 10-volt output from the MiniDAWN photodiode at 90 degrees to theincident light beam was split 1/11 and used as auxiliary detector inputfor the SP4500 Data Interface Module (0- to 1-volt A/D converter, ThermoSeparation Products). The voltage signals from both the ultravioletabsorbance detector and the static light scattering detector werecommunicated to and recorded by a personal computer and integrated bySP1000 software (Thermo Separation Products) to obtain peak areas inunits of millivolt-seconds (mV-sec). The ratio of the integratedabsorbance peak at 260 nm to the integrated absorbance peak at 280 nmfor the virus peak was consistently found to be approximately 1.2:1.Since the absorbance at 260 nm was consistently proportional to andhigher than that at 280 nm, the signal from the light scatteringdetector is compared to only the larger of these two absorbance signalsin the results summarized in Table 1.

TABLE 1 Integrated Integrated Ratio of Virus light 260 nm light Volumeparticles scattering absorbance scattering to Virus injected injected ×signal signal 260 nm dilution μL 10⁻⁹ mV-sec mV-sec absorbance 1/20 100.55 2,510 33.4 75 1/10 10 1.1 4,440 59.3 75 1/20 20 1.1 4,530 60.0 761/5 10 2.2 9,420 123 76 1/10 20 2.2 9,060 117 77 1/20 40 2.2 9,090 12374 1/5 20 4.4 18,980 239 80 1/10 40 4.4 19,540 241 81

FIG. 1 illustrates the results obtained for a 20 μL injection of a 1/5dilution of ONYX-015 (4.4×10⁹ particles). The difference in retentiontimes for the absorbance peak and the light scattering peak results fromthe volume of the capillary tubing between the two detectors, which isapproximately 0.125 mL (corresponding to 0.125 minutes at 1.0 mL/min).FIG. 2A compares the standard curves for the integrated light scatteringsignal and the integrated absorbance signal at 260 nm. FIG. 2B shows thesame data for the absorbance signal on an 80-fold expanded scale.

Measurements by both detection systems were linearly related to thenumber of particles injected and independent of the volume of dilutedsample that was injected within the tested range of 5.5×10⁸ to 4.4×10⁹particles and the range of 10 to 40 μL, as shown in Table 1. Resultsobtained with the two detection systems differed primarily in that theattenuated voltage signal from the light scattering detector was morethan 70-fold greater than the voltage signal from the ultravioletabsorbance detector.

EXAMPLE 2 Comparison of Light Scattering at a Right Angle and a LargerAngle

Proceeding as described in Example 1, light scattering signals werecollected at 90 and 138.5 degrees to the incident laser beam of aMiniDAWN light scattering detector with a 690-nm laser light source. The0- to 10-volt outputs from the photodiodes of the MiniDAWN werecollected by a personal computer using Astra software provided by WyattTechnology Corp. (Santa Barbara, Calif.). The 0- to 2-volt output fromthe 260-nm ultraviolet light absorbance detector was collected by apersonal computer using SP1000 software of the chromatographyworkstation (Thermo Separation Products) and the data from both dataacquisition systems were combined in an Excel workbook (MicrosoftCorporation, Seattle, Wash.). The results in FIG. 3 were obtained from asample containing 5.5×10⁸ adenovirus particles. For ease of comparisonof the results from the two light scattering detectors and theabsorbance detector, 3 volts were subtracted from the light scatteringsignal from the photodiode at 138.5 degrees and the ultravioletabsorbance signal was plotted on a 1,000-fold expanded scale. Inaddition, the time scale for the light scattering signals was correctedfor a delay of 0.125 minutes relative to the absorbance data (cf. FIG.1).

It is apparent from FIG. 3 that the peak heights and baselinefluctuations are similar for light scattering measured at 90 and at138.5 degrees and that the baseline of the absorbance signal varies farmore than do the baselines of the light scattering signals. Thesignal-to-noise ratio of the light scattering method in this example issuperior to that of the method described in Example 1. It is alsoapparent that this small quantity of virus (5.5×10⁸ adenovirusparticles) gives a peak height of more than one volt (i.e. 12% or 13% ofthe 10-volt dynamic range of the light scattering detectors), whereasthe same quantity of virus gives an absorbance peak height of slightlymore than one thousandth of a volt (1 mV), which is less than 0.1% ofthe 2-volt dynamic range of the absorbance detector. That a quantity ofvirus that produces an absorbance peak of only 0.0013 absorbance units(optical density units at 260 nm) at its maximum can yield a lightscattering signal of more than 10% of full scale is truly unexpected.

EXAMPLE 3 Sensitivity and Detection Limits

Portions of a suspension of adenovirus (ONYX-015, Onyx PharmaceuticalsRichmond, Calif.) containing 11.8×10¹⁰ particles/mL were seriallydiluted 2-fold through 128-fold to produce virus suspensions that rangedin concentration from 9.2×10⁸ particles/mL to 11.8×10¹⁰ virusparticles/mL (the undiluted sample). Injections of 50 μL of suspendingbuffer and of each dilution were chromatographed on a 1-mL Resource Qanion-exchange column (Amersham-Pharmacia Biotech, Piscataway, N.J.),essentially as described in Example 1. Proceeding as described inExample 1, light scattering signals were collected at 90 degrees to theincident laser beam of a MiniDAWN light scattering detector with a690-nm laser light source. The 0- to 10-volt output from the photodiodeof the MiniDAWN and the 0- to 2-volt output from the 260-nm ultravioletlight absorbance detector were communicated to and recorded by apersonal computer using Astra 4.00 software provided by Wyatt TechnologyCorp. (Santa Barbara, Calif.). With this data acquisition system, a peakwith the retention time of the virus was detected by light scattering inall of the dilutions of virus (9.2×10⁸ to 11.8×10¹⁰ particles/mL,containing 4.6×10⁷ to 5.9×10⁹ virus particles in the 50-μL samples). Incontrast, no discernable peak was seen by Astra analysis of theultraviolet absorbance signals in samples containing fewer than 7.4×10⁸virus particles, i.e. in the last four dilutions.

In accordance with a November 1996 recommendation of the InternationalConference on Harmonization of Technical Requirements for Registrationof Pharmaceuticals for Human Use (ICH, Geneva, Switzerland), the Limitof Detection (LOD) and Limit of Quantitation (LOQ) of a calibrationcurve are given, respectively, by 3.3 times and 10 times the ratio ofthe standard deviation of the response noise to its slope. In addition,the ICH defines the sensitivity of a test method as the slope of thecalibration curve. According to these definitions, the LOD and LOQ ofthe light scattering signal were calculated to be 0.8×10⁸ and 2.6×10⁸respectively. The detection limits were found to be several times higherfor the absorbance measurements than for the light scatteringmeasurements and the sensitivity of the light scattering method wasfound, in this example, to be approximately 3,000 times that of theabsorbance method, based on a peak height of 119 mV per 10⁸ virusparticles by light scattering (correlation coefficient, R²=0.995)compared with a peak height of 0.039 mV per 10⁸ particles by absorbanceat 260 nm (R²=0.982). While the Astra software is not well suited forquantitation of detector signals below 1 mV, the use of other dataanalysis applications and/or other devices, such as for output signalamplification, is within the scope of the present invention, as would beappreciated by those of skill in the art.

EXAMPLE 4 Determination of the Concentration of Purified Adenovirus

The concentration of a suspension of purified adenovirus inTris-buffered saline (pH 7.4) containing 1 mM MgCl₂ is measured bydetermining the intensity of scattered light after injectingapproximately 0.9 mL of the suspension into the flow-cell of a MiniDAWNlight scattering detector that has been calibrated by injection of aseries of dilutions of an adenovirus suspension of known concentration.The particle concentration that is measured in this batch mode issubstantially equal to the particle concentration that is measured bythe chromatographic methods described in Examples 1 and 2. An advantageof the method of this example, compared to measurement techniques thatrequire disruption of the virus particles (e.g. the methods of Maizel etal. supra or Mittereder et al. supra), is that the present exampleemploys a non-destructive test of the concentration of virus particles,permitting recovery of the virus suspension for further use. Inaddition, this batch method (which requires a larger sample volume thandoes a chromatographic method) can measure much lower concentrations ofviruses than can be measured with a size-exclusion chromatographicmethod.

EXAMPLE 5 Measurement of Adenovirus Concentration in Solution ContainingHuman Serum Albumin

The concentration of a suspension of purified adenovirus inphosphate-buffered saline (PBS, pH 7.4) containing 1% (w/v) human serumalbumin is measured by determining the intensity of scattered lightafter injecting 1-2 mL of the suspension into a MiniDAWN lightscattering detector that has been calibrated by injection of a series ofdilutions of an adenovirus suspension of known concentration. Theparticle concentration of this suspension could not be measured byprotein assay (Bistocchi et al. supra) or by ultraviolet absorbanceassay (Maizel et al. supra) because of the interference with thosemethods by the carrier protein; however, the intensity of lightscattering is hardly influenced by the carrier protein and the particleconcentration that is measured by this batch method is essentially thesame as is obtained with a chromatographic method (e.g. based on sizedifferences or charge differences) that separates the virus from theaccompanying carrier protein before measuring the intensity of lightscattering. Furthermore, the method of this example is more precise thana method for quantitation of virus particles that is based on thepolymerase chain reaction (PCR).

EXAMPLE 6 Quantitation of Virus in a Partially Purified VirusPreparation

A partially-purified preparation of virus from a process employed in themanufacture of a vector for use in gene-therapy (e.g., adenovirus) issubjected to fractionation by zone centrifugation in a sucrose gradient.The contents of the gradient are caused to flow through alight-scattering detector that has been calibrated by injection of aseries of dilutions of a virus suspension of known concentration. Theintensity of scattered light in the region of the gradient that containsthe virus, when compared to the calibration curve, indicates thedistribution of the concentration of the virus particles. Aftercollecting the virus-containing region of the density gradient, theconcentration of virus particles in the product pool is determined bythe method of Example 4.

It should be noted that the present invention is not limited to onlythose embodiments described in the Detailed Description. Any embodimentthat retains the spirit of the present invention should be considered tobe within its scope. The invention is only defined by the followingclaims.

What is claimed is:
 1. A method for determining the number orconcentration of virus particles in a sample, comprising the steps of:measuring the amount of light scattered by said virus particles with adetector placed away from the axis of illumination; and comparing theamount of light scattered to one or more known light scattering valuescorrelated to one or more known concentrations of the virus.
 2. Themethod of claim 1, wherein the comparing step comprises comparing theresults of the measuring step to a standard light scattering curvecomprising a plurality of data points, wherein said data points arebased on said light scattering values and said concentrations of thevirus.
 3. The method of claim 1, further comprising the step ofpurifying said virus particles using a fractionation system prior tosaid measuring step.
 4. The method of claim 3, wherein saidfractionation system includes a chromatographic medium.
 5. The method ofclaim 4, wherein said chromatographic medium comprises an ion-exchangemedium.
 6. The method of claim 4, wherein said chromatographic mediumcomprises a size-exclusion medium.
 7. The method of claim 4, whereinsaid chromatographic medium comprises an affinity medium.
 8. The methodof claim 3, wherein said fractionation system includes a componentselected from the group consisting of a chromatography column, acountercurrent distribution apparatus, a two-phase system, a gradient,and a centrifuge.
 9. The method of claim 1, wherein said virus is anadenovirus.
 10. The method of claim 1, wherein said virus is selectedfrom the group consisting of human herpesvirus, human papilloma virus,adeno-associated virus, flavivirus, dengue virus, Japanese encephalitisvirus, human T-cell lymphotrophic virus, hepatitis virus, humanimmunodeficiency virus (HIV), cytomegalovirus (CMV), Epstein-Barr virus,reovirus, vaccinia virus, parvovirus, feline leukemia virus, cauliflowermosaic virus and tomato bushy stunt virus.